Name: isolating myofibrils from skeletal muscle biopsies and determining contractile function with a nano-newton resolution force transducer
Uploaded: 2020-05-07T14:00:00
Description: Watch this Scientific Journal Video about Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer at JoVE.com

This project was funded by AFM-Telethon and A Foundation Building Strength for Nemaline Myopathies. The authors wish to acknowledge the creator of the products mentioned in this article, IONOptix Inc.

The protocol for obtaining human biopsies was approved by the institutional review board at VU University Medical Center (#2014/396) and written informed consent was obtained from the subjects. The protocol for obtaining animal muscle biopsies was approved by the local animal ethics committee at VU University (AVD114002016501)
1. Preparation and myofibril isolation
NOTE: Use previously described methods to glycerinate biopsies, prepare the different calcium concentrat...

<ol>
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Described is a protocol to assess the contractile function of myofibrils isolated from human or animal skeletal muscle tissues. The force resolution of this setup has been described before by Chavan et al.12. In short, it is determined by the random fluctuations of the length of the Fabry-P&#233;rot cavity formed between the detection fiber and the cantilever, which produce the dominant part of the noise at the output of the readout (expressed in V) that, multiplied by the deflection sensitivity (...

Striated muscle cells are indispensable for daily life activities. Limb movement, respiratory function, and the pumping motion of the heart rely on the force generated by muscle cells. Skeletal muscle consists of muscle fascicles containing bundles of single muscle fibers (Figure 1A). These muscle fibers are comprised of myofibrils, which are formed by serially linked sarcomeres (Figure 1B,D). The sarcomeres contain thin and thick filaments. These primarily consist of chains of actin and myosin molecules, respectively (Figure 1B). Actin-myosin interactions are responsible for the force-generating capacity of muscle. Patients with mutations in genes encoding for sarcomeric proteins, such as nebulin, actin, and troponin T, suffer from muscle weakness due to contractile dysfunction1.
The quality of muscle contractility can be studied at various levels of organization, ranging from in vivo whole muscles to actin-myosin interactions in in vitro motility assays. During the past decades, several research groups have developed setups to determine the contractility of individual myofibrils2,3,4,5,6,7,8,9,10. These setups are based on the detection of changes in laser deflection from a cantilever (i.e., optical beam deflection) caused by the contraction of the myofibril (for details, see Labuda et al.11). Although determining the contractile function of myofibrils has some limitations (e.g., the dynamics of the excitation-contraction coupling processes that are upstream of the myofibrils are lacking), there are multiple advantages to this approach. These include: 1) the ability to assess actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres; 2) the ability to assess actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils (when measuring the contractility of single muscle fibers ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility) (Figure 1D); 3) the small diameter of myofibrils (~1 &#181;m, Figure 2A) and the lack of membranes allow for almost instant calcium diffusion into the sarcomeres. Furthermore, if structural damage is present in the myofibrils, they likely break during their isolation or during the experiment. Hence, assessing myofibril contractility is an elegant method to study the basic mechanisms of muscle contraction and to understand whether disturbed actin-myosin interactions are the primary cause of muscle disease caused by mutations in sarcomeric proteins.
This protocol presents a newly developed setup to determine the contractility of myofibrils incorporating a cantilever force probe with nano-Newton resolution (i.e., Optiforce). This force probe is based on the principle of interferometry. Interferometry enables the use of relatively stiff cantilevers. This makes it possible to measure force with little deflection of the cantilever, approaching isometric contractions of the myofibril. The probe allows for the assessment of low passive and active forces that are produced by a single myofibril isolated from different muscle biopsies, including those from human subjects, with a high signal-to-noise ratio. The optical cantilever force probe incorporated in this setup is based on a Fabry-P&#233;rot interferometer12. The interferometer detects small displacements between an optical fiber and a gold-coated cantilever mounted on a ferrule (Figure 3). The gap between the optical fiber and the cantilever is called the Fabry-P&#233;rot cavity. Myofibrils are mounted between the probe and piezo motor using two glue-coated glass mounting fibers. The force produced by the myofibril can be mathematically derived from the interferometer data. Interferometry is based on the superposition or interference of two or more waves (in this setup three light waves). Laser light with a wavelength between 1,528.77&#8211;1,563.85 nm is emitted from the interferometer and is sent through the optical fiber. In the probe, the light is reflected 1) at the interface between the optical fiber and the medium (Figure 3A); 2) at the interface of the medium and the cantilever (Figure 3B); and 3) at the interface between the metal and gold coating of the cantilever (Figure 3C). The reflection at interface A and B is dependent on the refractive index (n) of the medium in which the probe is submerged. The light, consisting of the three superimposed reflections, returns to a photodiode in the interferometer. The photodiode measures the intensity of the light, which is the result of the interference pattern of the three superimposed reflections. When contractile force is generated by activating or stretching a myofibril, the myofibril pulls on the cantilever. This movement changes the cavity size (d) and consequently, the number of wavelengths that fit in the cavity. The light reflected at the cantilever will have a different phase, resulting in a different interference pattern. The photodiode records this change of interference pattern intensity as a change in Volts. Subsequently, myofibril force generation is calculated from this change, taking into account the cantilever stiffness. The force probe is calibrated by the manufacturer by pushing the tip of the mounting needle, attached to the free handing end of the cantilever, against a weighing scale while keeping the bending of the cantilever equal to a multiple of the wavelength of the readout laser13. Thus, interferometry is a highly sensitive method to detect small changes in distance, allowing for measurement of forces with nano-Newton resolution. This resolution enables the assessment of myofibrillar force production with a high signal-to-noise ratio. While traditional interferometry limits the range of measurements to the linear part of the interference curve, using a lock-in amplifier and modulation of the laser wavelength overcomes this limitation14. This is explained in more detail in the discussion section.
To measure myofibril active tension, a fast-step perfusion system was incorporated to expose the myofibril to calcium solutions (Figure 4A). The fast-step perfusion system enables solution changes to occur within 10 ms. Because of their small diameter, calcium diffusion into the myofibrils is nearly instantaneous. Hence, this system is particularly suitable for measuring the rates of actin-myosin binding during activation and release during relaxation. The rate of activation (kACT) and relaxation (kREL) can be determined from the activation-relaxation curves. Also, by exposing the myofibrils to calcium solutions of increasing concentration, the force-calcium relationship and calcium sensitivity can be determined.
Furthermore, a piezo length motor enables fast stretching and shortening of the myofibril. This offers the possibility to study the viscoelastic properties (i.e., passive tension) of the myofibril, as well as performing a rapid shortening and restretch of the myofibril to determine the rate of tension redevelopment (kTR). The parameters retrieved from both active and passive tension experiments can be altered by gene mutations in a sarcomeric protein.
This custom-built setup was used to measure the active and passive contractile characteristics of myofibrils isolated from healthy human, patient, and mouse skeletal muscle.

<table>
	<tbody>
		<tr>
			<td>Bio Spec Products, Inc.</td>
			<td>985370-XL</td>
			<td>To isolate myofibrils</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom coded</td>
			<td></td>
			<td>Matlab</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom fabricated</td>
			<td></td>
			<td>Includes Labview program to control over serial connection; To control valves</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom fabricated</td>
			<td></td>
			<td>To cool the Peltier module</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom fabricated</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Custom fabricated</td>
			<td></td>
			<td>Aluminum tissue chamber</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom fabricated</td>
			<td></td>
			<td>To control the valves; Includes PC software to control over USB</td>
			<td></td>
		</tr>
		<tr>
			<td>IonOptix</td>
			<td></td>
			<td>System controller software: data recording software with advanced signal generator for piezo and fast-step</td>
			<td></td>
		</tr>
		<tr>
			<td>IonOptix</td>
			<td>MCS100</td>
			<td>To record sarcomere length</td>
			<td></td>
		</tr>
		<tr>
			<td>IonOptix</td>
			<td></td>
			<td>Includes: Optiforce (interferometer), Micromanipulators, Signal interface, Piezo motor and controller. Based on the MyoStretcher</td>
			<td></td>
		</tr>
		<tr>
			<td>IonOptix</td>
			<td></td>
			<td>Force probe</td>
			<td></td>
		</tr>
		<tr>
			<td>Koolance</td>
			<td>ADT-EX004S</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Koolance</td>
			<td>EX2-755</td>
			<td>To cool the Peltier module</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft</td>
			<td></td>
			<td>Data registration</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus</td>
			<td>TH4-200</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma-Aldrich</td>
			<td>529265</td>
			<td>Poly(2-hydroxyethyl methacrylate); Coating for microscope slides to prevent sticking of tissue</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma-Aldrich</td>
			<td>78471</td>
			<td>Crystals to dissolve in ethanol resulting in glue</td>
			<td></td>
		</tr>
		<tr>
			<td>TE Technology, Inc.</td>
			<td>TE-63-1.0-1.3</td>
			<td>To cool the tissue flow chamber</td>
			<td></td>
		</tr>
		<tr>
			<td>TE Technology, Inc.</td>
			<td>TC-720</td>
			<td>Includes PC software to control over USB</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan Trading AG</td>
			<td>20736652</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan Trading AG</td>
			<td>20739263</td>
			<td>Syringe pump to induce backgroundflow together with fast-step perfusion system; Outflow from tissue flow chamber</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo scientific</td>
			<td>2441081</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warner Instruments (Harvard Bioscience, Inc.)</td>
			<td>Discontinued</td>
			<td>Alternative: SF-77CST/VCS-77CSP</td>
			<td></td>
		</tr>
		<tr>
			<td>Warner Instruments (Harvard Bioscience, Inc.)</td>
			<td>TG150-4</td>
			<td>To perfuse the tissue</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>1 PC for IonWizard and 1 PC for other software</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating myofibrils from skeletal muscle biopsies and determining contractile function with a nano-newton resolution force transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Data traces were recorded and opened with the system controller software (see Table of Materials). Complete traces or selected segments were exported to the clipboard or text file for further analysis with a desired software. Valves to control flow of the different solutions were switched with custom software or manually. A custom MATLAB script was used to analyze the rates of activation, tension redevelopment, and relaxation. The maximum active force and the peak and the plateau force of the passive for...

Watch this Scientific Journal Video about Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer at JoVE.com

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Assessing the contractile properties of myofibrils isolated from striated muscle can be used to determine whether sarcomere dysfunction is a primary cause of muscle weakness due to mutations in sarcomeric proteins. This technique can be used to acquire data with a very high signal-to-noise ratio with nanonewton resolution. This protocol is also suitable to measure contractility in permeabilized cardiomyocytes.Keep in mind that the force transducer is very fragile. So when removing the glue from the mounting needle or when gluing a myofibril, you need to have a steady hand, a lot of practice, and a lot of patience. Before mounting the tissue, place a homogenizer rod in the tube containing the muscle tissue.Keeping the tube on ice, spin the rotor for 15 seconds on speed five. At the end of the homogenization, transfer 50 microliters of the resulting myofibril suspension and 250 microliters of relaxing solution onto a poly-HEMA coated slide in a tissue bath. Cover the bath with a lid to protect the mixture from dust and wait 5 to 10 minutes to allow the myofibrils to sink to the bottom of the drop.During the sinking of the myofibrils, heat Shellac an ethanol glue at 65 degrees Celsius for 30 to 60 seconds before adding about six microliters of glue onto an uncoated glass slide. Repeatedly dip the tip of each mounting needle into the glue until a layer of glue is visible. Then use micro-manipulators to move the probe and piezo vertically to make room for the tissue bath on the microscope stage and remove the glass slide containing the glue.After mounting the myofibrils, to measure the sarcomere length, move the piezo and/or force probe to set the initial sarcomere length of the myofibril to 2.5 micrometers. Using the vessel function of the system controller software, measure the myofibril length and width. Use the microscope stage to position the myofibril in the center of the video image and stretch a rectangle from one side of the myofibril to the other, taking care to include the dark edge of the glue droplets.To start recording the data, click start. After five seconds, click pause. The length will have been recorded.To measure the width, rotate the camera 90 degrees to view the contrast of the edge of the myofibril itself. Adjust the rectangle and click start to start recording the data. After five seconds, click pause.The width will have been recorded. To position the theta glass, use the eyepiece and the manipulator to carefully move the theta glass toward the myofibril. Align the top channel of the theta glass with the myofibril and perform a fast-step to check the position.Turn on the background relaxing flow to check the theta glass alignment and use a Luer valve lever to turn on the inflow of the flow chamber. Then to start draining the flow chamber and to prevent overflowing of the flow chamber, set the outflow pump valve to bath valve two, the micro step mode to micro, the plunger target to 48, 000, and the plunger speed to 38 to 40. To measure the rate of tension redevelopment, calculate the piezo movement necessary to slacken the myofibril by 15%and enter this value into the signal generator.Click resume to continue recording the data and open valves one and six to start the flows of the relaxing solution and the different calcium concentrations through the theta glass respectively. Select reset range on the interferometer to reset the range of the interferometer so that the baseline force is zero volts. When the force trace is stable, perform the theta glass fast-step with a 100 micrometer step size.When the force plateau is reached, perform the shortening re-stretch with the piezo. An activation relaxation trace will be recorded. Click pause.To perform a step-wise stretch, click start and reset the range of the interferometer so that the baseline force is zero volts. Perform a step-wise stretch with the signal generator. When the stretch is finished, use the piezo to shorten the myofibril to the slack length.Then press pause and stop and save the data. Here, force traces of an active force experiment with a myofibril isolated from healthy human quadriceps muscle are shown. The myofibril was activated five times with solutions with varying calcium concentrations, with an average maximum force of all of the myofibrils of approximately 123 millinewtons per square millimeter.The construction of a force calcium concentration curve from the plateau forces reached during each activation in each of the five calcium curves allows calculation of the calcium concentration at 50%of the maximum force production. As illustrated in this representative analysis, the myofibrils can be treated with multiple compounds in a single experiment to answer additional questions about the myofibril type. The active force and sarcomere length can also be measured to allow calculation of the rates of redevelopment, activation and relaxation.The steep rise shown in the dotted red box is caused by both viscosity and elasticity. The plateau resembles only the elastic component. As viscosity resists strain linearly, the force dropped after the strain was removed.When positioning the theta glass, make sure you align it properly with the myofibril. Otherwise, the activating solution might come in between the optical fiber and the cantilever of the force probe and this causes artifacts to occur in your data. Also, the myofibril may not activate properly.This method of perfusion is also suitable to determine the muscle fiber type of the myofibril. For example, you can first perfuse the myofibril with a normal calcium solution followed by perfusion of that same solution, but then add a muscle fiber-type specific force-enhancing compound.

isolating-myofibrils-from-skeletal-muscle-biopsies-determining

Research

JoVE Journal

Biology

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...